Analysis of 3- and 6-linked sialic acids in mixtures of gangliosides usingblotting to polyvinylidene difluoride membranes, binding assays, and various mass spectrometry techniques with application to recognition by Helicobacter pylori

A convenient approach to analyze 3- and 6-linked sialic acids in mixtures o f biologically active gangliosides was developed. The procedure was adapted to work on small amounts of material and included parallel tests, which al lowed direct analysis of structure and activity. The initial step in the pr ocedure was separation of a mixture of gangliosides by thin-layer chromatog raphy (TLC) and blotting to a polyvinylidene difluoride membrane. The gangl iosides were then analyzed (a) by direct desorption from the membrane and f ast atom bombardment mass spectrometry (FAB MS), (b) by membrane-binding as say using the NeuAc alpha B- and NeuAc alpha 6-specific lectins from Maacki a amurensis and Sambucus nigra, respectively, and (c) by TLC binding assay with radiolabeled bacteria. All experiments were performed on a mixture of gangliosides from human leucocytes, which contained species with affinity f or the human gastric pathogen Helicobacter pylori. The procedure was used w ith good results for gangliosides with up to seven sugars per ceramide. A t hree-sugar ganglioside was identified as GM3 with ceramides composed of sph ingosine (d18:1) and 20:0, h20:0, and 24:0 fatty acids. The sequences of fo ur bands in the five-sugar region were consistent with sialylparagloboside (NeuAc alpha 3/6Gal beta 4GlcNAc beta 3Gal beta 4Grlc beta Cer). The cerami des were composed of d18:1 and 24:1 fatty acid in the first and third bands , and d18:1 and 16:0 fatty acid in the second and fourth bands from the top . The sialic acid was shown to be 3-linked in the upper two bands and B-lin ked in the lower two bands. The same distribution of sialic acid and cerami des but the sequence elongated with one N-acetyllactosamine unit was observ ed in the less resolved interval containing seven-sugar glycosphingolipids. The direct comparison of binding of lectins and radiolabeled bacteria show ed that H. pylori recognized 3-linked sialic acid only. These results were supported by a novel technique of analysis of the sialic acid linkage posit ion by trifluoroacetolysis and gas chromatography/MS. Direct membrane/FAB M S was ineffective for species migrating below the seven-sugar region on the TLC. In this case, the membranes were instead cut in bands and the ganglio sides extracted by methanol before analysis by FAB MS. (C) 1998 Academic Pr ess.